This invention relates to methods for inhibiting or treating vaginal atrophy.
Vaginal atrophy is a condition occurring in 75 to 85% of postmenopausal women. Vaginal atrophy is marked by a significant thinning of the mucosa of the vagina. Symptoms resulting from the abnormally thin vaginal mucosa include vaginal dryness, discomfort, itching, dyspareunia, infection, inflammation, ulcers, discharge, and bleeding. Urinary incontinence and urinary tract infections can also accompany vaginal atrophy.
The normal vaginal mucosa consists of a stratified squamous epithelium, which undergoes multiple changes throughout the life of a woman. During puberty, the vaginal epithelium is highly proliferative, thick, and contains abundant glycogen. After menopause, estrogen levels decrease resulting in a decreased glycogen content and general reduction in vaginal secretions. In post-menopausal women suffering from vaginal atrophy, the vaginal mucosa thins and the cellular make-up changes significantly. The thin vaginal mucosa characteristic of vaginal atrophy lacks maturation, i.e., it consists of numerous parabasal cells and little or no superficial and intermediate cells, resulting in further decreased glycogen deposits and a higher pH. This loss of lubrication and increased pH can lead to many of the symptoms associated with vaginal atrophy, e.g., increased susceptibility to infections, vaginal dryness, and dyspareunia.
Vaginal atrophy is caused primarily by an estrogen deficiency; the mucosa of the vagina is an estrogen sensitive tissue and a well-known target organ for estrogen. At the time of menopause, the levels of estrogen produced by the ovaries rapidly decrease. This decrease in estrogen has pronounced effects on the vagina causing a rapid acceleration in the natural process of atrophy. Estrogen replacement therapy is often beneficial in treating vaginal atrophy. The administration of exogenous estrogen can dramatically reverse the atrophic process by causing the vaginal epithelium to undergo proliferation and maturation, resulting in an increase in vaginal mucosal thickness. The administration of exogenous estrogen also influences glycogen deposits and vaginal acidity, which can reduce susceptibility to bacterial infections.
Many postmenopausal women are however unable to use estrogens due to medical contraindications such as a history of breast, endometrial, ovarian or cervical cancer and various hematological disorders. In addition, some postmenopausal women who would benefit from estrogen replacement do not receive replacement due to fears of estrogens in general or undesirable side effects such as nausea, breast tenderness, vaginal bleeding, and fluid retention. The treatment of vaginal atrophy in patients who do not use exogenous estrogen is a significant therapeutic problem; most of these women are forced to endure their symptoms due to the lack of effective treatment alternatives. Clearly, an effective and safe agent, which positively affects the underlying physiology and thus improves the qualitative aspects of vaginal properties in post-menopausal women, would be useful.
Parathyroid hormone (PTH) is an important regulator of calcium and phosphorus concentration in extracellular fluids. PTH is synthesized as a preprohormone and, after intracellular processing, is secreted as an 84 amino acid polypeptide. PTH release and synthesis are controlled principally by the serum calcium level; a low level stimulates and a high level suppresses both hormone synthesis and release. PTH, in turn, maintains the serum calcium level by indirectly or directly promoting calcium entry into the blood at three sites of calcium exchange; gut, bone, and kidney. PTH acts directly to raise extracellular calcium by its actions on bone and kidney and indirectly by increasing the production of 1,25(OH)2D3 to enhance intestinal calcium absorption. A variety of cells, including kidney cells, lymphocytes, and osteosarcoma cells, possess receptors for PTH. A variety of in vitro and in vivo tests have been developed to assay for PTH activity. These include the measurement of cyclic AMP production in isolated canine kidney membranes, osteosarcoma cells, and human fibroblasts. In addition, a multiresponse PTH assay has been developed to measure both agonist and antagonist properties of PTH analogs.
Cultured human keratinocytes also make a PTH related protein, known as PTHrP. PTHrP was isolated from a human lung cancer cell line, and full-length complementary DNA clones encoding it have been inserted into expression vectors used to produce the peptide in mammalian cells. The clones were found to encode a family of distinct peptide hormones, one of which is a peptide of 36 amino acids that has significant homology with PTH in the amino terminal region; of the first 16 residues of this protein, eight were found to be identical to human PTH. In addition to regulating extracellular calcium levels, PTHrP is also a potent regulator of cellular proliferation, differentiation, and death of many cell types.
The similar activity profiles of PTH and PTHrP can be explained by their interaction with a common receptor, the type I PTH/PTHrP receptor, which is expressed abundantly in bone and kidney. In both hPTH and hPTHrP, the region encompassing amino acids 15-34 contains the principal determinants for binding to the PTHPTHrP receptor. (The format xPTH (y-z) is used hereafter to identify the peptide, where x refers to the species (e.g. h for human and b for bovine), y refers to the starting amino acid in the PTH amino acid sequence and z refers to the ending amino acid.) Although these regions show only minimal sequence homology (only three amino acid identities), both hPTH(15-34) (SEQ ID NO: 7) and hPTHrP(15-34) (SEQ ID NO: 22) peptides can block the binding of either hPTH(1-34) or hPTHrP(1-34) (SEQ ID NOs: 5, 17) to the PTHPTHrP receptor. A type II PTH receptor was also identified in lymphocytes and keratinocytes, as well as in insulinoma and squamous carcinoma cells (Orloffet al., Endocrinology 186:3016-3028, 1995). Another additional receptor, the PTH-2 receptor, was also recently identified and shown to respond predominantly to PTH but not to PTHrP.
Structure function analysis of PTHPTHrP has facilitated the design of many peptides, which can function as either PTHPTHrPPTH-2 receptor agonists or PTHPTHrPPTH-2 receptor antagonists. Many of these peptides are described in the literature. For example, some of the known peptide agonists include bPTH (1-34), hPTH(1-34), [Nle8,18, Tyr34] bPTH (3-34)NH2, hPTHrP (1-34), hPTHrP (1-36), (SEQ.ID.NOs:4, 5, 13, 17, 18; see for reference Nussbaum et al., J. Prot. Chem. 4:391-406, 1985; Keutman et al., Endocrinology 117:1230-1234, 1985, Orloff et al., Endocrinology 186:3016-3028, 1995). Peptides which are known to have antagonistic functions include hPTH(7-34), [Nle8,18, Tyr34]bPTH (7-34)NH2, [Tyr34]bPTH (7-34)N2, hPTHrP(7-34), [Leu11,D-Trp12]hPTHrP(7-34)NH2, [Asn10Leu11]hPTHrP(7-34)NH2, and [Asn10,Leu11,D-Trp12]hPTHrP(7-34)NH2(SEQ.ID.NOs: 8, 14, 15, 19, 24-26; see for reference Nutt et al. Endocrinology 127:491-493, 1990; Doppelt et al., Proc. Natl. Acad. Sci. USA 83:7557-7560, 1986; and U.S. Pat. Nos. 6,362,163 and 5,527,772). These peptide agonists and antagonists have been used previously to induce or to block various PTHPTHrPPTH-2 receptor functions including proliferation, differentiation, and stimulation of cyclic AMP (cAMP) production.